With at least 510 members, human protein kinases constitute the largest family of potential drug targets that have drawn tremendous attention from both academic and industrial researchers due to the demonstrated key regulatory roles of protein phosphorylation in essentially all cellular processes. Graves & Krebs, Pharmacol. Ther. 82:111 (1999); Schenk & Snaar-Jagalska, Biochim. Biophys. Acta., 1449:1 (1999). It is estimated that approximately 30% of current research spending on drug development focuses on this enzyme family, particularly in the areas of cancer, diabetes and immune diseases. Several kinase inhibitors have been approved by the FDA as anti-cancer drugs (Herceptin®, Gleevec®, and Tarceva®), and at least thirty modulators of protein kinase activities are currently being tested in late stage clinical trials. In addition, inhibitors of a number of kinases that act on non-protein substrates are also being developed as drug candidates. Such kinases include, e.g., phosphatidylinositol-3-kinase, ceramide kinase, glucokinase, guanylate kinase, adenosine kinase, and polyphosphate kinase. Efforts in drug screening to identify additional kinase inhibitors are still continuing.
Because there are many validated kinase targets, it is desirable to have a universal kinase assay available that can be applied to a large number of, if not all, kinases. In this regard, the commercially available assays from Promega and Cambrex that are based on monitoring ATP-to-ADP conversion in the course of enzymatic phosphorylation are potentially applicable to all ATP-dependent kinases. However, a serious drawback of this approach is that it relies on the determination of substrate disappearance rather than product accumulation and, hence, is not suitable for the quantification of the initial rate of the enzymatic reactions (the most informative and reliable measure of enzyme activity) with high signal-to-background ratio.
Recently, two novel homogeneous assays for ADP that employ an ADP-specific aptamer or ADP-dependent ribozyme as detection reagents have been developed and their utility for measuring activity of protein kinases has been demonstrated. Srinivasan et al., Chem. Biol., 11:499 (2004). However, the signal-to-background ratio observed in such assays are very low, and as such, the general applicability of these techniques remains to be further tested. In addition, the assays also have the drawback of requiring radioactively labeled ATP and thus are associated with radioactive hazardous waste.
As interest in kinase-based drug development continues intensifying unabatedly, there is great need for highly sensitive and easy to use assays that can be applied to a broad range of kinases.